Modified Antibody

ABSTRACT

Recombinant antibody-based molecules that trigger both T-cell and B-cell immune responses are disclosed. The recombinant molecules are comprised by at least one targeting unit and at least one antigenic unit connected through a dimerization motif. Also disclosed are nucleic acid molecules encoding the recombinant antibody-based molecule and methods of treating multiple myeloma or lymphoma in a patient using the recombinant antibody-based molecules or the nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/730,776 filed on Oct. 12, 2017, which is a divisional of Ser. No.14/872,290 filed on Oct. 1, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/353,548 filed on Jan. 19, 2012, now U.S. Pat.No. 9,169,322, issued Oct. 27, 2015, which is a divisional of U.S.patent application Ser. No. 10/786,907 filed on Feb. 25, 2004, now U.S.Pat. No. 8,932,603, issued Jan. 13, 2015, which is a divisional ofInternational Application No. PCT/N02004/000051, filed Feb. 25, 2004,which claims priority back to U.S. provisional application No.60/450,134, filed Feb. 25, 2003.

FIELD OF THE INVENTION

The present invention relates to a recombinant human antibody-basedmolecule called Vaccibodies, which are able to trigger both a T cell andB cell immune response. More particularly, Vaccibodies by themselvesinduce such strong immune responses that adjuvants are not necessarilyrequired. The present invention also relates to a method of treatinge.g. multiple myeloma by means of the said Vaccibodies.

SEQUENCE LISTING

This application contains a sequence listing electronically submitted asan ASCII text file identified as file name: Seqlist_DVC.txt, creationdate: Sep. 29, 2015, size: 8 kilobytes. The material of the ASCII textfile is incorporated-by-reference.

BACKGROUND OF THE INVENTION

Multiple myeloma (MM) is a bone marrow cancer in which a single plasmacell clone has turned malignant and produces monoclonal immunoglobulin(Ig). MM patients have a very poor prognosis. Although high responserates and increased survival can be achieved using high dosechemotherapy followed by autologous or allogeneic stem cell grafting,the majority of patients relapse and few, if any, are cured.

Myeloma cells produce monoclonal Ig that is unique for each tumor andthus for each individual patient. Ig is composed of two identical heavy(H) and two identical light (L) chains. L and H chains have highlydiversified variable (V) regions, VL and VH. VL and VH together form theFv (fragment variable) that contains unique antigenic determinantscalled idiotopes (Id). Idiotopes collectively constitute the idiotype ofthe Fv (of the Ig in casu). Induction of an immune response against theidiotype, so called Id-vaccination is a promising strategy in treatmentof B cell lymphomas and MM (Bendandi, Gocke et al. 1999) (Tao and Levy1993) (Huang, Wu et al. 2004) (Hakim, Levy et al. 1996) (King,Spellerberg et al. 1998) (Biragyn, Tani et al. 1999; Biragyn, Ruffini etal. 2002), and both anti-idiotypic antibodies (Sirisinha and Eisen 1971;Hough, Eady et al. 1976) and Id-specific T cells (Lauritzsen, Weiss etal. 1994) may be of importance. However, Id is a weak self-Ag in itsoriginal context (as part of Ig). Therefore, for vaccine purposes, it isimportant to enhance the immunogenicity of Id.

T helper cells (CD4+ T cells) recognize their antigen (Ag) after it hasbeen processed through engulfment of foreign proteins by APC,proteolytic breakdown into peptide fragments that are loaded onto MHCclass II molecules and transported to the surface of the APC where thepeptide-MHC complex is presented to T cell receptors (TCRs) of CD4+ Tcells. Activated CD4+ T cells stimulate cytotoxic T cells (CD8+ T cells)and B cells with the corresponding Ag specificity, initiating a broadresponse against the original Ag. A major problem concerningId-vaccination of MM patients is that Id-specific CD4+ T cells in thesepatients, as extrapolated from experiments in mice [18], probably aretolerant to Id V-region determinants on the highly abundant myelomaprotein. MM patients who have undergone autologous stem celltransplantation (ASCT) may be in an advantageous phase forId-vaccination for the following two reasons: 1) relief from T celltolerance to myeloma protein Id and 2) development of new T cells thatcan respond to Id-vaccination.

Targeting of T cell epitopes to surface molecules on APC with Troybodies(Lunde, Munthe et al. 1999), which are equipped with a T cell epitopeincorporated in a loop in a constant Ig domain results in increased Tcell stimulation by a factor of 100-100000 (Lunde, Rasmussen et al.2001). However, Troybodies do not include the Ag in its nativeconformation, such as Fv, and are therefore restricted to induction of Tcell responses. Therefore, to induce an anti-Id B cell response andanti-Id Abs, it is necessary to include the complete Fv of the Mcomponent of the patient. As for induction of an anti-Id T cellresponse, an inclusion of the entire Fv will greatly increase the chanceof including idiotope sequences binding the patient's HLA-molecules,which is a prerequisite for activation of Id-specific T cells.

There have been several approaches for rendering idiotypes moreimmunogenic. Protein vaccination with complete Id+ immunoglobulins (Ig)fused with granulocyte-macrophage colony-stimulating factor (GM-CSF)(Tao and Levy 1993), or CD40 ligand (Huang, Wu et al. 2004) enhances thelevel of anti-Id antibodies and results in protection against B-celllymphoma in mice. However, scFv-GM-CSF was effective only when injectedas protein and not as a DNA vaccine (Hakim, Levy et al. 1996). On theother hand, DNA vaccination employing scFv fused to IL-1βdid inducetumor immunity (Hakim, Levy et al. 1996). In another approach, scFv hasbeen genetically fused with fragment C from tetanus toxin and deliveredas a DNA vaccine by intramuscular (i.m.) injection. This strategy hasresulted in increased levels of anti-Id antibodies, Id-specific CD4+responses and protection against lymphoma a myelomas in mice. Themechanism of adjuvant activity of tetanus toxoid fragment C is unknown(King, Spellerberg et al. 1998). In a similar approach, scFv has beenfused to chemokines like MCP3, IP10 mDF2β(Biragyn, Tani et al. 1999;Biragyn, Ruffini et al. 2002) and has been used both as a DNA and asprotein vaccine (Biragyn, Tani et al. 1999). In several of thesestudies, foreign T cell epitopes corresponding to TT fragment-C orunique fusion sequences could have contributed to responses. Heightenedanti-Id antibody responses and tumor protection has been observed. Themechanism of action of scFv-chemokine is unknown. One possibility isthat the chemokine moiety targets Fv to chemokine receptors on APC forenhanced delivery of scFv. Alternatively, chemokines attract APC to thesite of injection. However, both the Fragment C and chemokine fusionstrategies rely on monovalent binding to their target molecules (King,Spellerberg et al. 1998; Biragyn, Tani et al. 1999). This is of concernbecause crosslinking has been shown to be of importance for optimalstimulation of T cells, e.g. for Troybodies (Lunde, Munthe et al. 1999).

With these considerations in mind, the inventors have designed a noveltype of recombinant antibody-like molecules called Vaccibodies, adivalent molecule comprising a flexible hinge, with no FcR binding andthat contain the Ag in its native conformation, with the purpose ofinducing both strong Id-specific Ab and T cell responses. Vaccibodiesare large and complex macromolecules, but, surprisingly, cells were ableto produce and export intact molecules.

SUMMARY OF THE INVENTION

The present invention relates to a novel type of human recombinantantibody-like molecules useful in the treatment of i.e. multiplemyeloma. These molecules, called Vaccibodies, bind APC and are able totrigger both T cell and B cell immune response. Moreover, Vaccibodiesbind divalently to APC to promote a more efficient initiation of animmune response. Hence, a major purpose of the present invention is toinduce a strong immune response to render adjuvants redundant.Vaccibodies comprise a dimer of a monomeric unit that consists of a scFvwith specificity for a surface molecule on APC, connected through ahinge region and a Cγ3 domain to a scFv in the COOH-terminal end; thelatter being of B cell lymphoma or myeloma origin (FIG. 1 ), althoughany origin is possible due to the cassette cloning system in theexpression vector. The said molecule is capable of inducing an immuneresponse against multiple myeloma, but extension to a generalvaccination strategy for any polypeptid should be feasible. The presentinvention also relates to a DNA sequence coding for this recombinantantibody based molecule, to expression vectors comprising these DNAsequences, cell lines comprising said expression vectors, to treatmentof mammals preferentially by immunization by means of Vaccibody DNA,Vaccibody RNA, or Vaccibody protein, and finally to pharmaceuticals anda kit comprising the said molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description and the accompanying drawings, wherein:

(FIG. 1 ) The structure of the Vaccibody. The two scFvs in white targetthe Vaccibody to surface molecules on APC. They may be replaced by othertargeting molecules, e.g. chemokine receptors. The hinge providesflexibility of the relative orientation of the two NH2-terminal scFvsand disulfide bridges the monomers. The CH3 domains (light grey) act asa spacer between the NH₂—and COOH terminal scFvs and participate in thedimerization through hydrophobic interactions. These dimerization motifsmay be replaced by other dimerization or multimerization domains. Thetwo scFvs shown in dark grey are the antigenic moiety of the Vaccibody.These scFvs are derived from the M component, thus harboring idiotypicsequences (black). The antigenic scFv may be replaced by any polypeptidederived from an antigenic source, conferring vaccine strategies towardsany antigen.

(FIG. 2 ) Principle. The Vaccibody is targeted to surface molecules onAPC, the complex is taken up by receptor-mediated endocytosis, processedand Id-peptides are presented to CD4+ T cells on MHC class II.Simultaneously, the Id may stimulate B cells with an anti-Id BCR. TheseB cells will also serve as APC for the CD4+ T cells. Thus, T and B cellswill cooperate to enhance the response, as indicated. Anti-Id B cellswill as a consequence develop into plasma cells that produce anti-Idantibodies.

(FIG. 3 ) The MHC class II-specific Vaccibodies bind CD19+ splenocytes(white). The NIP-specific Vaccibodies (negative controls—grey), do notbind to the splenocytes. Binding was detected with streptavidinPE and9A8bio which is a rat mAb that binds the antigenic Fv of M315 origin(Vλ½).

(FIG. 4 ) The NIP-specific control Vaccibodies exhibits binding to thehapten NIP. Supernatant from cells transfected with genes encodingNIP-specific Vaccibodies was added to ELISA plates coated with NIP-BSA.9A8 bio (antiVλ½) was used as the secondary Ab. Similar results wereobtained with a biotinylated anti IdAb (Ab2.1-4 bio).

(FIG. 5 ) The Vaccibodies exhibit binding to DNP, hence the antigenicscFv is correctly folded. The M315 Ab, from which the antigenic scFv isderived, is specific for the hapten DNP. ELISA plates were coated withDNP-BSA. Supernatants from cells transfected with genes encoding variousVaccibodies were added. Binding was detected with 9A8 bio.

(FIG. 6 ) APC pulsed with titrated amounts of MHC class II specificVaccibodies stimulated polarized M315-specific T cells fromTCR-transgenic SCID mice >100-1000 fold better than the NIP-specific,untargeted control Vaccibodies. There were no significant differencesbetween the Vaccibodies with a long sequence prior to the firstdisulfide bridge in the hinge (h1+h4) compared to the Vaccibodies with ashort hinge sequence above the first disulfide bridge (h4).

(FIG. 7 ) The MHC class II-specific Vaccibodies induce a strong anti-IdAb response in the absence of adjuvant. BALB/c mice were injected withVaccibodies, 20 μg and 200 μg, respectively. Blood samples were taken ondifferent time points for sera analysis. Shown is data from sera takenon day 28, 14 days after the second immunization of Vaccibodies. The MHCclass II-specific Vaccibodies induced a strong anti-Id Ab response. TheVaccibodies with the longest hinge (h1+h4) induced the strongest anti-IdAb response reaching 3-4 μg/ml in sera.

(FIG. 8 ) Construction of the two hinge-Cγ3 variants derived from hIgG3by PCR. The templates were from pUC19 containing modified hIgG3 constantregions were the h4 exon were connected to the CH3 domain (A) or the h1exon were connected to the h4 exon further connected to the CH3 domain(B) (Olafsen T et al, 1998). The primers inserted Hindlll (5′) and Sfil(3′) restriction enzyme sites. The hinge and CH3 domain are connected bya triplicate of the amino acids GlyGlyGlySerSer.

(FIG. 9 ) Construction of the hinge-Cγ3 segments derived from mIgG2b.The hinge and the CH3 genes were amplified from a pUC18 vectorcontaining the constant region of mIgG2b by PCR with two primersencoding a HindIII (5′) and a Sfil (3′) restriction enzyme site. The twoPCR fragments were joined by PCR SOEing. In this reaction, the hinge andCH3 domain were connected by a triplication of the amino acidsGly-Gly-Gly-Ser-Ser.

(FIG. 10 ) Construction of the scFv derived from the myeloma proteinM315. The cDNA that functioned as a template in the PCR reactions werederived from mRNA extracted from MOPC315.4 cells. The V regions werejoined by PCR SOEing resulting in a scFv. In this reaction, the Vregions were connected by a triplicate of GlyGlyGlyGlySer. Furthermore,the gene fragments encoding the complete scFv were flanked by Sfil andSall restriction enzyme sites.

(FIG. 11 ) Joining of the hinge-Cγ3 segments and the M315 scFv by PCRSOEing. This reaction introduced the Sfil site 5′ of the antigenic scFvencoding region.

(FIG. 12 ) Subcloning of the hinge-Cγ3-M315 scFv into pUC19. Threedifferent dimerization motifs were included, derived from mIgG2b orIgG3. In all cases, they consisted of hinge followed by a triplicate ofGlyGlyGlySerSer and CH3. Two different hinges were derived from hIgG3,one consisting of h1 linked to h4, and one consisting of h4, only.

(FIG. 13 ) Removal of two inconvenient BamHI restriction enzyme siteswithin the gene fragment encoding the antigenic scFv by QuickChange PCR.

(FIG. 14 ) Introduction of stop codon, a Sfil and a BamHI restrictionenzyme site downstream of the coding region by QuickChange PCR.

(FIG. 15 ) Subcloning into the C cassette of the expression vectorpLNOH₂ on Hindlll-BamHI.

(FIG. 16 ) Cloning of the V regions specific for NIP and MHCII. The Vregions were amplified and joined by PCR soeing resulting in scFvs. Thelinker connecting the V regions consists of a triplicate ofGlyGlyGlyGlySer. The gene fragments encoding the complete scFvs areflanked by Bsml/Munl and BsiWl sites. Linkers and restriction sites wereintroduced in the PCR reactions.

(FIG. 17 ) Subcloning into the expression vector pLNOH2 on Bsml/Munl andBsiWl.

(FIG. 18 ) The final Vaccibody construct.

(FIG. 19 ) Detailed figure of Vaccibody gene construct. The targetingunit is inserted between the Bsml/Mfel and BsiWl restriction enzymesites (The V cassette of the pLNOH₂ vector). The hinge-Cγ3-Fv315 isinserted between the HindIII and BamHI sites into the C cassette ofpLNOH2. The hinge and the Cγ3 domain as well as the two scFv's areconnected with (G4S)₃ linkers (black boxes). The Cγ3 and the Fv³¹⁵ areconnected through a GLSGL linker. The Fv³¹⁵ is inserted between twononidentical Sfil restriction enzyme sites. The antigenic unit anddimerization motif may be of any origin appropriate. Also, functionalfragments of Cγ3 may be employed, or a sequence which is substantiallyhomologous to the Cγ3 sequence or Cγ3 fragments.

(FIG. 20 ) Vaccibodies are secreted as functional molecules. Twodistinct Vaccibodies were tested, one carrying the hapten specificFv^(NIP) as targeting unit, while the other carried the MHC classII-specific Fv^(I-E) as targeting unit. Both carried the scFv from M315(Fv³¹⁵) as antigenic unit. a) 10% SDS-PAGE of metabolically labeledVaccibodies immunoprecipitated from culture supernatants oftransfectants with or without reduction of disulfide bonds bymercaptoethanol (ME). b) DNP-specificity of the Vaccibodies was measuredby ELISA. Supernatants from NSO cells transfected with Vaccibodies wereadded to ELISA plates coated with DNP-BSA. Data are illustrated as meanof triplicates and error bars indicate SEM. c) NIP-specificity wasmeasured by ELISA. ELISA plates were coated with NIP-BSA. Vaccibodies inboth b) and c) were detected by either 9A8-bio (αVλ½) or Ab2.1-4(specific for Id of Fv³¹⁵) Fv³¹⁵ carries Vλ2 and FvNIP carries Vλ1 andwill both bind 9A8 mAb. Only Fv³¹⁵ will bind Ab2.1-4 mAb. Fv^(I-E)carries Vκ and will bind neither of the mAbs.

(FIG. 21 ) Production of Vaccibodies by intramucular injection of nakedDNA plasmids followed by in vivo electroporation. Serum samples werecollected on day 14. Vaccibody plasmids were injected into I-E^(d)positive BALB/c mice, which were subsequently electroporated. a) Levelof Vaccibodies in sera was measured by ELISA, with DNP-BSA and 9A8bio asdescribed previously. b) The same day 14 sera samples were analyzed foranti-Id antibodies by ELISA. Microtiter plates were coated with M315 and187-bio (anti-mouse κ Ab) was used for detection. c) Comparison ofdetectable Vaccibody levels and anti-Id antibodies. The amount ofdetectable Vaccibodies in sera is shown on the y-axis and the level ofαld-Abs is shown on the x-axis.

(FIG. 22 ) Tumor avoidance. BALB/c mice were immunized once with nakedplasmids encoding MHC class II specific Vaccibodies (Fv^(I-E) Fv³¹⁵),nontargeted NIP-specific Vaccibodies (Fv^(NIP) Fv³¹⁵) or 0.9% NaCl byi.m. immunization into the two quadriceps muscles (25 μg/muscle)followed by in vivo electroporation. They were challenged with 1.6×10⁵MOPC315.4 myeloma cells s.c. and the first day tumor take were recorded.A tumor of >3 mm was scored as positive tumor take.

(FIG. 23 ) Induction of protective immunity against the MOPC315.4plasmacytoma. BALB/c mice were immunized once with naked plasmidsencoding MHC class II specific Vaccibodies (Fv^(I-E) Fv³¹⁵), nontargetedNIP-specific Vaccibodies (Fv^(NIP) Fv³¹⁵) or 0.9% NaCl by i.m.immunization into the two quadriceps muscles (25 μg/muscle) followed byin vivo electroporation. They were challenged with 1.6×105 MOPC315.4myeloma cells s.c. and their survival were compared.

(FIG. 24 ) Level of M315 myeloma protein in sera of mice on a) day 18and b) day 24 after MOPC315.4 challenge in BALB/c mice vaccinated i.m.with Vaccibody plasm ids followed by in vivo electroporation. M315 insera samples were measured by ELISA coated with anti-Id-mAb (Ab2.1-4)and detected by biotinylated anti-IgA mAb (8D2).

(FIG. 25 ) Chemokine Vaccibodies are secreted as functional molecules.MIP-1 a Fv³¹⁵ has mouse macrophage inflammatory protein 1 a as thetargeting unit and scFv from M315 (Fv³¹⁵) as the antigenic unit.Functionality of MIP-1α in Vaccibody format was measured in ELISA.Supernatants from 293E cells transfected with Vaccibodies were added toELISA plates coated with anti-mouse MIP-1α mAb (R&D Systems) anddetected by 9A8-bio (αVλ½).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiment(s) is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses.

The present invention relates to a recombinant human antibody-basedmolecule, called Vaccibodies, comprising dimers of a monomeric unit thatconsist of a single chain fragment variable (scFv) of immunoglobulins(Ig) with specificity for surface molecules on Ag presenting cells(APC), connected through a hinge region and a Cγ3 domain to a scFv inthe COOH-terminal end, the latter being derived from a myeloma protein(FIG. 1 ), although any origin is possible due to the cassette cloningsystem of the expression vector. The hinge region and the Cγ3 domains(carboxyterminal C domain of Ig) contribute to dimerization of theVaccibody through disulfide bridges in the hinge and strong hydrophobicinteractions between the two Cγ3 domains. Hence, the dimeric productwill include two preferably identical scFvs with binding specificity forthe same surface molecules on APC (FIG. 2 ), enabling bivalent binding.Bivalent binding (i.e. crosslinking) is of importance to triggeractivation of the target cell and thereby initiation of an immuneresponse. Also, bivalent binding provides increased binding strength dueto avidity effects, and increases the likelihood of receptor mediatedendocytosis into the APC and subsequent degradation inside the APC.Furthermore, the bivalent binding may provide important receptormediated intracellular signaling to the APC. The scFvs with a targetingfunction are either derived from B cell hybridomas expressing monoclonalantibodies (mAbs) that bind to surface molecules on APC, or they may bederived from any source, e.g. phage display libraries. The use of scFvsfrom B cell hybridomas as the targeting moiety opens for a great rangeof possible targets due to the large collection of B cell hybridomasthat produce mAbs which bind different surface molecules on APC.Furthermore, one may choose the nature of the signal given to thetargeted cell by employing agonistic or antagonistic mAbs. Growingknowledge of Ab-Ag interactions will allow the improvement of thebinding affinity of such mAbs to their Ag by amino acid replacements inthe binding sites. This can be performed by ordinary site-directedmutagenesis. For vaccine purposes, an attractive approach is to targetthe Vaccibodies to surface molecules expressed exclusively on subsets ofdendritic cells (DC) that are able to initiate a strong, specific immuneresponse towards the patients own Id. Examples of such target surfacemolecules on APC are CD40, Toll-like receptors and chemokine receptors.Because the targeting scFv is inserted into the V cassette of theexpression vector pLNOH2 (Norderhaug, Olafsen et al. 1997), it is easilyexchanged with other scFvs (FIG. 17 ).

The crucial dimerization motifs in the Vaccibodies constructed in theexamples so far, include hinge regions and Cγ3 domains. The hingecontributes to the dimerization through the formation of interchaindisulfide bridges. In addition, it functions as a flexible spacerbetween the domains allowing the two scFvs with targeting tasks to bindsimultaneously to two target molecules expressed with variable distances(FIG. 2 ). The C□3 domains contribute to the dimerization throughhydrophobic interactions. These dimerization motifs can be exchangedwith other multimerization moieties (e.g. from other Igisotypes/subclasses).

The C-terminal scFv derived from the monoclonal Ig produced by myelomaor lymphoma cells, also called the myeloma/lymphoma M component, can begenetically exchanged with other scFvs or any antigen because the vectorhas been constructed with a Sfi I restriction site (FIG. 8 ). Therefore,the scFv derived from the model myeloma protein used in the presentexample is easily swapped with scFv from the M component of any patientwith B cell lymphoma or multiple myeloma. Thus, this vector will allowfor rapid construction of individual patient specific vaccines. Thedimeric structure of the Vaccibody not only affords crosslinking, butshould also allow double loading of the patient Fv to the APC perVaccibody molecule compared to a single scFv combined to a singletargeting moiety. Furthermore, there is in a single Vaccibodyduplication of serological idiotypic determinants, which might be ofimportance for the anti-Id B cell response. The Vaccibodies lack a CH2domain and hence all FcR binding sites, and should therefore exclusivelybe taken up through its target molecules, such as MHC class II in theexample used and not by any FcRs, ensuring that a large proportion ofthe vaccine will arrive at the intended target cells. This is incontrast to vaccines that exert their effect through the binding to anFcR on a target cell (Ravetch and Bolland 2001).

Vaccibodies can be extended to a general medical treatment throughinduction of an immune response against any polypeptide of any origin.It is possible to replace the idiotypic scFv with other antigenicsequences of sufficient length to allow proper folding of thepolypeptide. This sequence may be derived from other cancer proteins orinfectious agents. Insertion of such a sequence in a Vaccibody formatmight also lead to activation of both arms of the immune response,similar to the Vaccibodies that are described herein, which comprise theidiotypic scFv. Immunization by means of Vaccibody protein, VaccibodyDNA, or Vaccibody RNA, the latter two executed e.g. by intramuscularinjection followed by electroporation (See Examples), are all feasiblemethods.

The scFvs on the NH2- terminal end of the Vaccibodies target theVaccibodies to APC through binding to surface molecules (FIG. 2 ), andin the example shown they bind to MHC class II. MHC class II isexpressed on all professional APC, so the Vaccibodies described hereinare able to target B cells, DC and macrophages. Targeting ofconventional Ag-Ab complexes to MHC class II induces activation ofspecific CD4+ T cells (Snider and Segal 1987; Casten and Pierce 1988).Targeting of Troybodies to MHC class II has previously been shown toenhance Ag presentation and T cell activation in vitro as well as invivo (Lunde, Western et al. 2002). In the antigenic region of themolecule, the Vaccibodies of the examples contain the scFv of themyeloma protein M315 derived from the BALB/c plasmacytoma MOPC315.4. TheA2315 light chain of M315 harbors three defined somatic mutations in theCDR3 loop and functions as a model idiotypic T cell epitope in a welldefined system (Bogen, Malissen et al. 1986; Bogen and Lambris 1989).

The Vaccibodies have been genetically assembled, and the DNA transfectedinto NSO cells, 293E cells and Cos-7 cells. Transfectants produce andsecrete the recombinant Vaccibody molecules. The targeting scFvs at oneend of the Vaccibodies exhibit binding to MHC class II (FIG. 3 ). Theantigenic scFv at the other end of the Vaccibody binds DNP(di-nitro-phenyl, the specificity of M315) (FIG. 5). Thus, both thetargeting and antigenic scFvs retain the same folding pattern as intheir original context. We have evidence that Vaccibodies have theability to induce strong T cell responses through their binding to APCand presentation of Id-peptides on class II molecules to Id-specificCD4+ T cells in vitro (FIG. 6 ). Furthermore, since they have intact Fvof the M-component, they elicit anti-Id antibodies in significantamounts in vivo when injected into BALB/c mice without adjuvants (FIG. 7).

To determine if MM patients treated with ASCT achieve remission with alow serum myeloma protein concentration, ELISA should performed for eachpatient's myeloma protein because routine assays (agarose gelelectrophoresis, combined with immunofixation) have only a sensitivityof about 0.2-1 mg/m I, which is far too insensitive. The kinetic data ofthe serum myeloma protein levels will indicate if and whenId-vaccination may best be performed post ASCT to avoid the problem of Tcell tolerance of newly educated thymic emigrants. To achieve this, miceare immunized with DNA encoding patient specific Vaccibodies by in vivoelectroporation of muscle cells. Sera from immunized mice are absorbedon anti human Ig-Sepharose to remove crossreactive antibodies andthereafter eluted to obtain purified highly Id-specific antibodies.Sandwich ELISAs specific for each patient's myeloma are performed asfollows: The purified anti-Id Ab from mice is coated in wells. Serumfrom the patient in casu is added. Myeloma protein binding to anti-Idantibodies will be detected by use of Ab specific for human IgG or IgA.The sensitivity of such sandwich ELISAs is usually <1 ng/ml, whichis >106 times more sensitive than routine assays. Furthermore, tomonitor development of new T cells, profile of T cells in blood will bemonitored by flow cytometry with Vβ-specific mAbs, in combination withother markers.

The present invention relates to a pharmaceutical comprising the abovedescribed recombinant based antibody, DNA/RNA sequences, or expressionvectors according to the invention. Where appropriate, thispharmaceutical additionally comprises a pharmaceutically compatiblecarrier. Suitable carriers and the formulation of such pharmaceuticalsare known to a person skilled in the art. Suitable carriers are e.g.phosphate-buffered common salt solutions, water, emulsions, e.g.oil/water emulsions, wetting agents, sterile solutions etc. Thepharmaceuticals may be administered orally or parenterally. The methodsof parenteral administration comprise the topical, intra-arterial,intramuscular, subcutaneous, intramedullary, intrathekal,intraventricular, intravenous, intraperitoneal or intranasaladministration. The suitable dose is determined by the attendingphysician and depends on different factors, e.g. the patient's age, sexand weight, the kind of administration etc. The present invention alsorelates to a kit comprising Vaccibody DNA, RNA, or protein fordiagnostic, medical or scientific purposes.

The above described nucleotide sequences may preferably be inserted intoa vector suited for gene therapy, e.g. under the control of a specificpromoter, and introduced into the cells. In a preferred embodiment thevector comprising said DNA sequence is a virus, e.g an adenovirus,vaccinia virus or an adeno-associated virus. Retroviruses areparticularly preferred. Examples of suitable retroviruses are e.g.MoMuLV or HaMuSV. For the purpose of gene therapy, the DNA/RNA sequencesaccording to the invention can also be transported to the target cellsin the form of colloidal dispersions. They comprise e.g. liposomes orlipoplexes.

In a preferred embodiment of the present invention naked Vaccibody DNAconstruct is injected intra-muscularly into mice, whereupon the site ofinjection is subject to in vivo electroporation. This DNA vaccinationresulted in production of Vaccibody protein which conferred life-savingprotective immunity on a majority of the mice.

MATERIALS AND METHODS

Mice

BALB/cABom were from Bomholtgaard (Ry, Denmark). The λ2315-specificTCR-transgenic mice on a BALB/c background (Bogen, Gleditsch et al.1992)were bred in our animal facility.

Cell Lines

The 14-4-4S Hybridoma (Ozato, Mayer et al. 1980) and NSO cells werepurchased from American Type Culture Collection (ATCC, Manassas, Va.).293E cells, a variant of the 293 cell line expressing the Epstein-Barrvirus EBNA1 protein.

Construction of Vaccibodies

The gene for the hIgG3 hinge and CH3 domain was cloned from the pUC19vector containing hinge genetically combined with Cγ3 genes of the hIgG3subclass (Olafsen, Rasmussen et al. 1998). Two variants of the hingelength in the humanized Vaccibodies were made; one with just the h4 exonconnected to the CH3 domain (sh) and one with both exon h1 and h4connected to the CH3 domain (1h) (FIG. 8 ). The primers includedrestriction enzyme sites (underlined):

5′h4: tag caa gct tgg cca gcg cag gga g;3′CH3: cag gcc acc gag gcc ttt acc cgg aga cag ggaThe h1 exon were introduced directly upstream of the h4 exon byQuickChange PCR using these primers Qh1a: ctccaatcttctctctgca gag ctcaaa acc cca ctt ggt gac aca act cac aca gag ccc aaa tct tgt gac ac andQh1b: gt gtc aca aga ttt ggg ctc tgt gtg agt tgt gtc acc aag tgg ggt tttgag ctc tgcagagagaagattgggag.

The murine Vaccibodies have a complete hinge and CH3 domain of themIgG2b subclass picked up by PCR from a pUC18 vector containing the Cγ2bgenes (FIG. 9 ). The primers included restriction enzyme sites(underlined) or linkers (bold) with the complementary sequences(italic):

5′hinge: tagcaagctt ca gag ccc agc ggg ccc; 3′hinge:5′tcc acc tcc gct gct tcc acc gcc tgg gca ttt gtg aca ctc ctt g; 5′CH3:gga agc agc gga ggt gga agt gga ggg cta gtc aga gct cca ca; 3′CH3:cag gcc acc gag gcc acc cgg aga ccg gga gat g.

The hinge and the CH3 domain were then joined by PCR SOEing.

The Antigenic V region genes were cloned from the plasmacytoma MOPC315.4(Eisen, Simms et al. 1968). The V regions were obtained by extractingmRNA from the MOPC315.4 cell line with oligo (dT)-coated magneticDynabeads (Dynal). First strand cDNA were then made and used as templatefor PCR amplification of the V region genes using specific primersannealing to the exact ends of the M315 V region sequences. The primersincluded restriction enzyme sites (underlined) or linkers (bold) withthe complementary sequences (italic). The primer sequences were:

5′VH: ggc ctc ggt ggc ctg gat gta cag ctt cag gag tca; 3′VH:gcc aga gcc acc tcc gcc aga tcc gcc tcc acc tga gga gac tgt gag agt ggt;5′VL: ggc gga ggt ggc tct ggc ggt ggc gga tcg cag gctgtt gtg act cag gaa; 3′VL: gacg tcgac tag gac agt gac ctt ggt tcc.The VH and VL genes were then joined by PCR soeing to a scFv format(FIG. 10 ).

The complementary sequences in the tags 3′ of the Cγ3 coding region and5′ of the M315 VH coding region enabled the M315 scFv to be combinedwith the three different hinge-CH3 genes by PCR SOEing (FIG. 11 ). Theproducts of this reaction were then digested with HindIII and Sall andsubcloned into a pUC19 vector (FIG. 12 ). Two BamHI restriction enzymesites inside the V regions of M315 were removed by QuickChange PCR (FIG.13 ) using primers: BamHI VL1:at gcc aac tgg ata caa gaa aaa cc; BamHIVL2: gg ttt ttc ttg tat at cca gtt ggc at, BamHI VH1: tgg aac tgg atacgg cag ttt cc and BamHI VH2: gg aaa ctg ccg tat cca gtt cca. Afollowing QuickChange PCR using primers:

3′VL stop1: gtc act gtc cta tga ggcctgcagggcc ggatcc gtcgactctag and3′VL stop2: cta gag tcg ac ggatcc gaccctacagacc tca tag gac agt gac,were then performed to introduce a stop codon (bold), a Sfil and a BamHIrestriction enzyme site (underlined) downstream of the coding region(FIG. 14 ).

The final construct is then digested with HindIII and BamHI andsubcloned into the expression vector pLNOH₂ (FIG. 15 ) (Norderhaug,Olafsen et al. 1997).

The V region genes providing specificity for MHC class II had previouslybeen cloned from the 14-4-4S hybridoma (Lunde, Western et al. 2002),which produces an Ab specific for the Eα chain (determinant Ia.7) of theI-E MHC class II molecule (Ozato, Mayer et al. 1980). Specific primersannealing to the exact ends of the V region sequences with tags designedto include restriction enzyme sites (underlined) or linker sequences(bold) with the complementay sequences (italic). The primer sequenceswere: 5′VL: gac att caattg aca cag tct tct cct gct tcc; 3′VL: gcc agagcc acc tcc gcc aga tcc gcc tcc acc ttt gat ttc cag ctt ggt gcc; 5′VH:ggc gga ggt ggc tct ggc ggt ggc gga tcg cag gtc cag ctg cag cag t; 3′VH:ga cataca actcacc tga gga gac ggt gac tga gg. The V region genes givingspecificity for the hapten NIP (Neuberger 1983) were designed with thesimilar tag sequences except for the 5′VL primer: 5′VL: ggtg tacattcccag gct gtt gtg act cag gaa; 3′VL: gcc aga gcc acc tcc gcc aga tcc gcctcc acc tag gac agt cag ttt ggt acc t; 5′VH: ggc gga ggt ggc tct ggc ggtggc gga tcg cag gtc caa ctg cag cag cc; 3′VH: ga cataca a ctc acc tgagga gac tgt gag agt ggt. The VL and VH were then joined by PCR SOEing(FIG. 16 ) and subcloned into the V cassette pLNOH₂ vector containingthe hinge-CH3-scFvM315 genes (FIG. 17 and FIG. 18 ). Likewise, other Vgenes conferring a desired specificity are isolated from hybridomas orfrom phage selected from phage display libraries. They are then PCRamplified using primers designed in the same manner as above andsubcloned after PCR SOEing in the targeting-cassette (FIGS. 17 and 18 ).Rearranged V_(H) and VK genes conferring specificity for HLA-DP were PCRamplified from cDNA from the 22C1 hybridoma, which produces an antibodywith pan HLA-DP specificity. The V genes were reamplified with newprimers containing sites for direct cloning into the expression vectorspLNOK and pLNOH₂ (Norderhaug and Olafsen, 1997);

5′-VL, ggtgtgcattccgacattgtgctcacc; 3′-VL,cgtacgttctactcacgttttatttccagct; 5′-VH, gtgcattccgaggtgcagctgcaggagtct;3′-VH, cgtacgactcacctgaggagaccgtagc.Furthermore, scFV was generated by PCR SOEing using the followingprimers:

5′VL, g gtg tgcattc cga cat tgt gct cac c 3′VL:gcc aga gcc acc tcc gcc aga tcc gcc tcc acc gtt tta ttt cca gct 5′VH:ggc gga ggt ggc tct ggc ggt ggc gga tcg gag gtg cag ctg cag gag tct3′VH, cgtacg act cac ctg agg aga ccg tag c

In 3′VL and 5′VH the sequences in bold+ italics are complementary andantiparallell, thus hybridising to generate the gene fragment encodingthe linker region. Anti CD14 V regions are cloned from the mousehybridoma 3C10 (ATCC).

The chemokine genes were cloned from thioglycolate stimulated peritonealmacrophages. 4m1 2% thioglycolate were injected i.p. into Balb/c mice. 3days later peritoneal macrophages were collected and mRNA was extractedwith oligo (dT)-coated magnetic Dynabeads. First strand cDNA was madeand used as template for PCR amplification of chemokine genes (RANTESand MIP-1α) using specific primers: 5′MIP-1α: ggtg tacattc cgc gcc atatgg agc tga cac, 3′MIP-1α: ga cataca act cac ctg cat tca gtt cca ggt cagtg 5′RANTES: ggtg tacattc c gcc tca cca tat ggc tcg g 3′RANTES: gacataca a ctc acc tga cat ctc caa ata gtt gat gta ttc. The differenttargeting unit genes were then digested with Munl and BsiWl or Bsml andBsiWl, respectively and subcloned into the V cassette pLNOH₂ vectorcontaining the hinge-CH3-scFvM315 genes (FIG. 17 and FIG. 18 ). CD40ligand is cloned from T cells that are activated with LPS for 4 hoursbefore mRNA is extracted for preparation of cDNA. The cDNA is used astemplate in a PCR reaction with primers specific for the CD40 ligandsequence. Furthermore, this sequence is reamplified with primersdesigned to facilitate subcloning in the targeting cassette as describedabove.

Production and Purification of Vaccibodies

The pLNOH₂ vector carrying the Vaccibody genes was transfected into NSOcells by electroporation, and supernatants from single coloniesresistant to 800 μg/ml G418 were analyzed for Vaccibody secretion after2-3 weeks, using ELISA. DNP-BSA was used as coat, and biotinylatedrat-anti mouse Vλ½ (9A8-bio) was used for detection. The NIP-specificVaccibodies were additionally screened in an ELISA using NIP-BSA ascoat. The cells selected for high Vaccibody production were grown inRollerbottles (VWR) and affinity purified from supernatant using acolumn made by DNP-lysine (Sigma) coupled to fast flow Sepharose. TheVaccibodies were eluted with 0.05M DNP-glycine (Sigma) and theflow-through was run on an ion-exchange Cl— Dowex 1×8 resin column(Sigma). The eluted Vaccibodies were dialyzed against PBS/0.05% NaN3 andsterile PBS, before the vaccibody concentration were calculated fromabsorbance values at 280 nm.

Ab and Flow Cytometry

Ab and reagents used for flow cytometry were 9λ8 biotin, FGK.45 biotin,streptavidin PerCP, anti-CD19 PE and anti-mIgG2a PE (BD Pharmingen).BALB/c spleen cells were double stained with anti-CD19 PE andVaccibodies. Bound Vaccibodies were detected by 9λ8-bio and streptavidinPerCP. Twenty thousand cells were run on FACSCalibur (BD Biosciences,Mountain View, Calif.) and analyzed using the WinMDI software.

Metabolic Labelling and Immunoprecipitation

2×10⁶ cells were labelled for 6h at 37° C. in RPMI lacking methionine,cysteine (BioWhittaker) containing 100 μCi³⁵[S]-methionine, cysteine(Amersham). The SN was harvested and immunoprecipitated with ratanti-mouse Vλ½ (9λ8) on a wheel ON at 4° C. 10 μl Dynabeads coated withsheep anti-rat IgG (Dynal AS, Oslo, Norway) were incubated with theprecipitate for 1 h on a wheel and the Dynabeads were collected with aDynal Magnetic Particle Concentrator rack (Dynal MPC). The beads werewashed three times in ice cold PBS with 1% NP40 and resuspended in 10 μl1× sample buffer. The proteins were eluted from the beads by incubatingthe samples at 95° C. for 3 minutes. The Vaccibodies were run on a 10%SDS-PAGE gel, with a 5% stacking gel, at 40 mA for 1h, using a BIO RADminiprotean II gel electrophoresis apparatus. The gels were subsequentlyfixed in 30% methanol and 10% acetic acid for 30 minutes prior to 30 minincubation with Amplify (Amersham), before drying and exposing toBIOMAX-MR film (Eastman Kodak Company, Utah, USA).

T Cell Proliferation Assay

Irradiated (2000rad) BALB/c splenocytes (5×10⁵cells/well) were used as asource of APC. Titrated amounts of different MHC CLASS II— andNIP-specific Vaccibodies were added to the splenocytes. A 91-107 λ2³¹⁵synthetic peptide were used as a positive control. The assays were putup in 150 μl cultures in 96-well flat-bottom microtiter wells andincubated for 4 h at 37° C. (Lunde, Western et al. 2002). The cultureswere then washed three times before addition of 200 μl polarizedλ2³¹⁵-specific Th2 cells (2×10⁴) derived from TCR transgenic SCID mice.After 48h, the cultures were pulsed for 16-24h with 1 μCi³[H] dThd. Thecultures were harvested and, and incorporated ³[H] dThd was measuredusing a TopCount NXT scintillation counter (Packard, Meriden, Conn.).

In Vivo Experiments

BALB/c mice were injected subcutaneously (s.c) with 200 μg or 20 μgpurified Vaccibody proteins in PBS on day 0, 14 and 28. Blood sampleswere taken on day 14 and 28 before revaccination and then on day 35, 42and 49, before they were sacrificed according to the Humane End Pointprocedure.

Measurement of Antibody Responses

Anti-idiotypic Abs against M315 were measured by ELISA. The wells werecoated with 2 μg/ml M315. Anti-Id Ab in the sera were detected by abiotinylated anti-mouse Vκ Ab (187.1 bio), anti-mouse IgG1 bio oranti-mouse IgG2a bio (both from BD Pharmingen). Ab2.1-4 (an anti-Id mAbthat bind λ2³¹⁵) was used as standard.

Vaccination

Protein vaccination: BALB/c mice were injected subcutaneously (s.c) inthe right flank region with 20 μg or 200 μg purified class II— or NIPspecific Vaccibodies (F_(v) ^(1-E)F_(v) ³¹⁵,F_(v) ^(NIP)F_(v) ³¹⁵) inPBS on day 0 and 14. PBS was injected as negative control. Blood sampleswere collected from the leg vein on different time points after the lastimmunization. Anti-idiotypic antibodies with specificity withspecificity for Fv³¹⁵ were measured by ELISA. The wells were coated with2 μg/ml M315. Anti-Id Ab in the sera were detected by a biotinylatedanti-mouse κ mAb (187.1 bio). Ab2.1-4 (an anti-Id mAb binding Fv³¹⁵(Kristoffersen, Hannestad et al. 1987) was used as standard.

DNA Vaccination and Electroporation

Five to ten weeks old Balb/c mice were purchased from Bomholtgaard (Ry,Denmark). The animals were anaesthetized by intraperitonal injectionwith 9 g Pentobarbital/mice and the legs were shaved. Conductive gel wasapplied at the skin and 50 μl vector DNA diluted in 0.9% NaCl, wasinjected into the quadriceps. Following injection, electroporation wasperformed, by applying rod electrodes to the skin near the site of theinjection and subjecting the site to an electrical potential comprising10 trains of 1000 pulses each, with a pulse length at two times 200 Sec(positive 200Sec and negative 200 Sec) with 600 s interval between eachpulse and with a current limit of 50 mA (about 150-174 V/cm) (Tollefsen,Tjelle et al. 2002).

Blood samples were collected from the leg vein on different time pointsand heart puncture was performed on the day they were sacrificed. Serumsamples were analyzed for the presence of correctly folded Vaccibodies.The ELISA was performed with DNP-BSA as coat and 9λ8-bio as detected Ab,as described above. In addition, serum samples were analyzed for anti-IdAbs by ELISA as described above.

Tumor Challenge

Protein Vaccibodies-MOPC315.4: BALB/c mice (6-10 weeks old) wereinjected s.c. with 160 μg class II-or NIP-specific Vaccibodies in PBS inthe right flank region on day 0 and 14. On day 28, 1.6×10⁵ MOPC315.4cells were injected s.c. on the right flank. Mice were inspected twiceweekly. Tumor size development was monitored by palpation and use of acaliper. A tumor of 3 mm in diameter was scored as tumor take. Mice werekilled when tumor size reached 20 mm with no sign of tumor necrosis.

DNA Vaccibodies-MOPC315.4: DNA vaccination was performed at day 0 asdescribed above. On day 14, 1.6×10⁵ MOPC315.4 cells were injected s.c.in the right flank region. Tumor size development was monitored bypalpation and use of a caliper. The mice were sacrificed when the tumorsize reached 20 mm. Blood samples were collected on different timepoints from the leg vein. Levels of M315 myeloma protein in sera werequantified in a sandwich ELISA with Ab2.1-4 as coat by biotinylatedanti-Cα (8D2) mAb as detection Ab. Tumor size, tumor take, survivalcurves and statistical analyses were calculated by use of Graph PadPrism 3.0 software (San Diego, Calif.).

EXAMPLES

By way of example the following experiments demonstrate that Vaccibodiesbind APC and are able to trigger both T cell and B cell immune response.Moreover, the following experiments show that Vaccibodies induce astrong immune response rendering adjuvants redundant. The experimentsdemonstrate that said molecule is capable of inducing an immune responseagainst multiple myeloma and, further, the feasibility of treatment ofmammals by immunization by means of Vaccibody DNA or Vaccibody protein.The experiments also demonstrate that another attractive approach is totarget the Vaccibodies to surface molecules expressed exclusively onsubsets of dendritic cells (DC), like e.g. chemokine receptors. Thefollowing examples are meant to illustrate how to make and use theinvention. They are not intended to limit the scope of the invention inany manner or to any degree.

Example 1

Vaccibodies are produced and secreted as functional dimerized moleculesand is itself bound by the anti-Vλ½ antibody (9λ8) (Bogen 1989) and theanti-idiotypic antibody Ab2.1-4 (Lauritzsen, Weiss et al. 1994).

The M315 mAb binds the hapten di-nitro-phenyl (DNP) (Eisen, Simms et al.1968). Therefore, to verify that Vaccibodies were produced, secreted andcorrectly folded as functional molecules, the antigenic units ofVaccibodies were tested in ELISA for their capability to bind DNP, 9λ8and Ab2.1-4 mAbs. FIG. 20 b shows that both the NIP-specific and theMHCII-specific Vaccibodies, that both have scFv³¹⁵, bind DNP, 9λ8 andAb2.1-4. This was the case with all Vaccibodies containing scFv³¹⁵, boththe ones with long human dimerization unit, short human dimerizationunit and murine dimerization unit (FIG. 5 ). We next tested thetargeting units of the Vaccibodies. These were found to be correct;first, the NIP-specific Vaccibodies bound NIP-BSA in ELISA (FIG. 4 andFIG. 20 c ), while the MHCII-specific Vaccibodies did not (FIG. 20 c ).Second, the MHCII-specific Vaccibodies bound to I-E expressing BALB/csplenic B cells (H-2d) as detected by flow cytometry, whereas theNIP-specific Vaccibodies did not (FIG. 3 ). To check for correcthomodimerization, the Vaccibodies were metabolically labelled by growthof transfected cells in medium containing ³⁵S-methionine, vaccibodieswere immunoprecipitated from supernatant using specific antibodies, andanalyzed by SDS-PAGE. As would be expected from the theoreticalconsideration of FIG. 2 , both the F_(v) ^(NIP) F_(v) ³¹⁵ and F_(v1-E)F_(v) ³¹⁵transfectomas secreted dimeric Vaccibodies of ˜130 kDa. Afterreduction of disulfide bonds, the Vaccibodies are degraded to monomericchains of ˜65 kDa (FIG. 20 a ).

Example 2

MHC class II-specific Vaccibodies enhance λ2³¹⁵-specific stimulation ofCD4+ T cells.

Class II-specific and non-targeting NIP-specific Vaccibodies were mixedwith antigen presenting cells (APC) and compared for their ability toinduce specific T cell activation. Irradiated BALB/c splenocytes wereused as APC. The BALB/c strain has the H-2^(d) haplotype, hence theyexpress I-E^(d) molecules necessary for both targeting of the MHCII-specific Vaccibodies and presentation of the λ2³¹⁵ epitope tospecific CD4+ T cells.

The APC were pulsed with the different Vaccibodies for 4h andsubsequently washed. Washing was performed to reduce the chance thatI-E^(d)-specific Vaccibodies in the culture medium could diminish T cellstimulation by blocking I-E^(d) molecules (Lunde, Western et al. 2002).Polarized Th2 cells from mice transgenic for a λ2³¹⁵-specific I-E^(d)restricted TCR (Lauritzsen, Weiss et al. 1993) were added as responder Tcells. The dose response curve in FIG. 6 shows that the λ2³¹⁵ epitopewas presented 100-1000 times more efficiently to TCR-transgenic Th2cells when they were carried in the APC-targeted MHC II-specificVaccibodies (both the ones with short and long human dimerization units)compared to the non-targeted NIP-specific Vaccibodies. It should benoted that Vaccibodies do not include an FcγR binding site; hence theNIP-specific Vaccibodies should not be able to enter cells viareceptor-mediated endocytosis.

Example 3

Level of anti-idiotypic antibodies in sera of mice that receivedVaccibodies as proteins in saline s.c. in the absence of adjuvant.

In the protein vaccination protocol, BALB/c mice were immunized twice,spaced two weeks apart, with 20 or 200 μg MHC II— specific Vaccibodiesor NIP-specific Vaccibodies in PBS. Note that no adjuvant was employed.Sera from immunized mice taken at various time points after the secondvaccination were then analyzed for anti-idiotypic antibodies bindingM315 in ELISA. The MHC II-specific Vaccibodies elicited significanthigher anti-idiotypic antibody responses after 14 days after the secondimmunization than did NIP-specific Vaccibodies. Vaccibodies with a longhuman dimerization unit induced best anti-idiotypic Ab responses (FIG. 7). Thus targeting of Vaccibodies enhanced anti-Id immuneresponses,however, by this route of immunization, also the non-targetedVaccibodies induced some responses.

Example 4

Protein Vaccibodies detected in serum after injection of DNAintramuscularly and in vivo electroporation.

It has recently been described that skeletal muscle can produceantibodies after injection of Ig genes and electroporation (Tjelle2004). We therefore investigated if functional Vaccibodies were producedby i.m. plasmid injection and electroporation. Since the F_(v) ^(I-E)F_(v) ³¹⁵ Vaccibodies are specific for I-E^(d) molecules present inBALB/c (H-2d), these Vaccibodies should be rapidly absorbed by theI-E^(d) positive cells in BALB/c. By contrast, the non-targeted Fv^(NIP)F_(v) ³¹⁵ Vaccibodies should not be absorbed. Indeed, 14 days after asingle injection of 50 μg Vaccibody plasmid in quadriceps andelectroporation, F_(v) ^(NIP) F_(v) ³¹⁵ Vaccibody protein was detectedin significant amounts in serum, while there was no detectable F_(v)^(I-E) F_(v) ³¹⁵ (FIG. 21 a ).

Example 5

Anti-Id antibodies in serum after injection of Vaccibody DNAintramuscularly and electroporation.

Analysis of the same day 14 sera samples for anti-idiotypic antibodiesdemonstrated that mice i.m. injected/electroporated with the MHC classII —targeted F_(v) ^(I-E) F_(v) ³¹⁵ Vaccibody DNA, had developedantibodies that bound idiotypic Fv from the MOPC315.4 tumor (FIG. 21 b). This result was in distinct contrast to the lack of anyanti-idiotypic antibody response in mice injected with the non-targetedF_(v) ^(NIP) F_(v) ³¹⁵ Vaccibody DNA (FIG. 21 b ). Taken together withthe results described in example 4, the results demonstrate a formidableeffect of targeting to MHC class II (I-E^(d)) positive cells fordevelopment of a strong humoral response. Control mice injected i.m.with 0.9% NaCl followed by electroporation had neither Vaccibodies noranti-idiotypic Abs in day 14 sera (FIG. 21 a-c ).

Example 6

Induction of protective immunity against the MOPC315.4 myeloma:Vaccibody DNA injection/electroporation.

Intramuscular vaccination with MHC class II-targeted F_(v) ^(I-E) F_(v)³¹⁵ Vaccibody plasm ids and subsequent electroporation induced strongprotection against a challenge with MOPC315.4 myeloma cells, p<0.001,compared to control mice injected with 0.9% NaCl and electroporated(FIG. 23 ). By contrast, non-targeted F_(v) ^(NIP) F_(v) ³¹⁵ Vaccibodyplasmid immunization was ineffective compared to the saline controlgroup, p=0.2739 (FIG. 23 ). The appearance of tumor was delayed in micevaccinated with F_(v) ^(I-E) F_(v) ³¹⁵ compared to F_(v) ^(NIP) F_(v)³¹⁵ (FIG. 22 ). One of the F_(v) ^(I-E) F_(v) ³¹⁵ vaccinated micedeveloped a tumor of maximum 6 mm (day 20) in diameter that regressedand was completely undetectable from day 28 (data not shown). Thepresence of M315 myeloma protein in sera confirmed the tumor sizemeasurements (FIG. 24 ). These results show that protection against theMOPC315.4 myeloma can be achieved by i.m. DNA vaccine followed byelectroporation and that the protection requires targeting of thetumor-derived scFv to MHC class II (I-E^(d)) positive cells.

Example 7

Chemokines are functional as targeting units in the Vaccibody format

Supernatant from cells transfected with Vaccibody construct with MIP-1αin the targeting unit, long human dimerization unit and M315 scFv in theantigenic unit, were collected and tested in ELISA for binding to ananti-mouse MIP-1α mAb and 9λ8 bio. The Vaccibodies containing MIP-1αbound to anti-MIP-1α mAb, while the NIP-specific Vaccibodies did not(FIG. 25 ).

Example 8

The chemokine RANTES is functional as targeting unit in the Vaccibodyformat

In the same manner, a vector with a gene encoding Vaccibodies like thosedescribed in example 7 was produced, with the exception that thetargeting unit was the mouse chemokine RANTES. Supernatant from cellstransfected with this construct was collected and tested in ELISA forthe presence of Vaccibodies. The experiment showed that this Vaccibodyvariant was expressed and exported as a functional molecule.

Example 9

Flaggelin as targeting unit in the Vaccibody format.

In the same manner, a vector with a gene encoding Vaccibodies like thosedescribed in example 7 was produced, with the exception that thetargeting unit was flaggelin. Supernatant from cells transfected withthis construct will be collected and tested in ELISA for the presence ofVaccibodies.

Example 10

Soluble CD40 ligand as targeting unit in the Vaccibody format

In the same manner, a vector with a gene encoding Vaccibodies like thosedescribed in example 7 was produced, with the exception that thetargeting unit was soluble CD 40 ligand from the mouse. Supernatant fromcells transfected with this construct will be collected and tested inELISA for the presence of Vaccibodies.

Example 11

Anti-Toll-like-receptor 2 as targeting unit in the Vaccibody format.

In the same manner, a vector with a gene encoding Vaccibodies like thosedescribed in example 7 was produced, with the exception that thetargeting unit was a scFv with specificity for toll-like-receptor 2 fromthe mouse. Supernatant from cells transfected with this construct willbe collected and tested in ELISA for the presence of Vaccibodies.

Example 12

Anti-CD14 is functional as targeting units in the Vaccibody format

In the same manner, a vector with a gene encoding Vaccibodies like thosedescribed in example 7 was produced, with the exception that thetargeting unit was scFv with specificity for human CD 14. Supernatantfrom cells transfected with this construct was collected and tested inELISA for the presence of Vaccibodies. The results showed that thisVaccibody variant was expressed and exported as a functional molecule.

Example 13

Anti-HLA-DP is functional as targeting units in the Vaccibody format.

In the same manner, a vector with a gene encoding Vaccibodies like thosedescribed in example 7 was produced, with the exception that thetargeting unit was scFv with specificity for HLA-DP. Supernatant fromcells transfected with this construct was collected and tested in ELISAfor the presence of Vaccibodies. The results showed that this Vaccibodyvariant was expressed and exported as a functional molecule.

Example 14

Tuberculosis antigen in the Vaccibody antigenic cassette.

A nucleic acid encoding a tuberculosis antigen (cattle antigen) will beinserted into the antigenic unit of the Vaccibody construct.

Example 15

Telomerase antigen in the Vaccibody antigenic cassette

hTERT, an antigenic region of the telomerase ribonucleoprotein, will beinserted into the antigenic unit of the Vaccibody construct.

Example 16

HIV Gp120 antigenic in the Vaccibody antigenic cassette

A nucleic acid encoding a gp120 derived molecule will be inserted intothe antigenic unit of the Vaccibody construct.

Example 17

Vaccibodies with patient specific scFv of myeloma origin in theantigenic cassette.

This study has been initiated, but has not yet been completed.

Bone marrow aspirate from patients suffering from multiple myeloma canbe collected. The mononuclear cells (MNC) can be separated using adensity gradient solution of Ficoll-Isopaque (Lymphoprep™ fromAxis-Shield PoC AS). Total RNA can be isolated (TRIzol® Reagent fromInvitrogen™ Life Technologies) from MNC, and cDNA can be made from mRNA(First-Strand cDNA Synthesis Kit from Amersham Biosciences (Not I-d(T)18bifunctional primer)). This cDNA can be used as template in PCR withprimers that amplify the V genes of the heavy or light chain of themultiple myeloma lg. The sense primers are family specific and localizedin the leader regions (VH1-7, VK1-6 and VL1-10), and the anti-senseprimers are localized in the first part of the C regions (one primereach for IgG, IgA, kappa and lambda). PCR products can be ligated into avector (pGEM®-T Easy Vector from Promega), and transformed into E. coli.DNA samples isolated from individual colonies can be sequenced. Gettingthe same sequence from three different colonies originating from threedifferent PCRs confirms that the V regions from the myeloma Ig have beenisolated. PCR SOEing can be performed and reamplification is done withprimers including tags with sites for Sfil as described in FIG. 19 . Forone patient such primers had the sequence:

5′TAVH 5′ ACGTAGGCCTCGGTGGCCTGCAGATCACCTTGAAGGAGTCT 3′TAVK5′GATCCGGCCCTGCAGGCCTCATTTGATCTCCAGCTTGGTCCC

The resulting vector can be transiently transfected into 293E cells.Supernatants can be tested in ELISA for the presence of suchVaccibodies. They can also be injected into BALB/c mice. The presence ofanti-Idiotypic antibodies can be measured in ELISAs against serum fromthe mice and serum from the patients.

All references cited herein are incorporated in their entireties byreference.

The description of the invention is merely exemplary in nature and,thus, variations that do not depart from the gist of the invention areintended to be within the scope of the invention. Such variations arenot to be regarded as a departure from the spirit and scope of theinvention.

REFERENCES

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1. A method of treating multiple myeloma or lymphoma in a patient inneed thereof, the method comprising administering to the patient, arecombinant antibody-based molecule comprising two targeting units andtwo antigenic units connected through a dimerization motif, or a nucleicacid encoding said recombinant antibody-based molecule. 2-6. (canceled)7. The method of claim 1, wherein at least one targeting unit is aligand.
 8. (canceled)
 9. The method of claim 7, wherein said ligand is achemokine.
 10. The method of claim 9, wherein said chemokine is RANTESor MIP-1α. 11-13. (canceled)
 14. The method of claim 1, wherein thetargeting units have the ability to target antigen presenting cells(APC). 15-17. (canceled)
 18. The method of claim 1, wherein theantigenic unit(s) is/are an antigenic scFv.
 19. The method of claim 18,wherein the antigenic scFv is derived from a monoclonal Ig produced bymyeloma or lymphoma. 20-26. (canceled)
 27. The method of claim 1,wherein the dimerization motif comprises a hinge region and animmunoglobulin domain.
 28. (canceled)
 29. The method of claim 27,wherein the hinge region has the ability to form one or several covalentbonds.
 30. (canceled)
 31. The method of claim 27, wherein theimmunoglobulin domain is a carboxyterminal C domain, or a sequence thatis substantially homologous to said C domain.
 32. (canceled)
 33. Themethod of claim 27, wherein the immunoglobulin domain has the ability tohomodimerize. 34-35. (canceled)
 36. The method of claim 1, comprisingadministering the nucleic acid to the patient to induce production ofthe recombinant antibody-based molecule.
 37. (canceled)
 38. Arecombinant antibody-based molecule comprising two targeting units andtwo antigenic units connected through a dimerization motif, or a nucleicacid encoding said recombinant antibody-based molecule. 39-42.(canceled)
 43. The recombinant molecule of claim 38, wherein at leastone targeting unit is a ligand.
 44. (canceled)
 45. The recombinantmolecule of claim 43, wherein said ligand is a chemokine.
 46. (canceled)47. The recombinant molecule of claim 45, wherein said chemokine isMIP-1α. 48-57. (canceled)
 58. The recombinant molecule of claim 38,wherein at least one antigenic unit is derived from a bacterium.
 59. Therecombinant molecule of claim 58, wherein the bacterium derivedantigenic unit(s) is/are a tuberculosis antigen.
 60. The recombinantmolecule of claim 38, wherein at least one antigenic unit is derivedfrom a virus. 61-75. (canceled)
 76. A pharmaceutical compositioncomprising a recombinant molecule of claim 38 and a physiologicallyacceptable diluent or carrier. 77-82. (canceled)